[go: up one dir, main page]

WO2011097577A2 - Compositions et procédés pour traiter ou prévenir une dégénérescence de la rétine - Google Patents

Compositions et procédés pour traiter ou prévenir une dégénérescence de la rétine Download PDF

Info

Publication number
WO2011097577A2
WO2011097577A2 PCT/US2011/023925 US2011023925W WO2011097577A2 WO 2011097577 A2 WO2011097577 A2 WO 2011097577A2 US 2011023925 W US2011023925 W US 2011023925W WO 2011097577 A2 WO2011097577 A2 WO 2011097577A2
Authority
WO
WIPO (PCT)
Prior art keywords
valproic acid
valpromide
lithium
vpa
cell death
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2011/023925
Other languages
English (en)
Other versions
WO2011097577A9 (fr
Inventor
Shalesh Kaushal
Syed Mohammed Noorwez
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Florida
University of Florida Research Foundation Inc
Original Assignee
University of Florida
University of Florida Research Foundation Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University of Florida, University of Florida Research Foundation Inc filed Critical University of Florida
Publication of WO2011097577A2 publication Critical patent/WO2011097577A2/fr
Publication of WO2011097577A9 publication Critical patent/WO2011097577A9/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
    • A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/14—Alkali metal chlorides; Alkaline earth metal chlorides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents

Definitions

  • Age-related macular degeneration is a chronic disease associated with aging that gradually destroys central vision.
  • the macula consists of millions of densely packed cones and rods.
  • AMD Age-related macular degeneration
  • several small drusen or a few medium-sized drusen are detected on the macula in one or both eyes by ophthalmascope examination.
  • ophthalmascope examination At the intermediate stage, many medium-sized drusen or one or more large drusen are detected. This accumulation is typically characterized by perceptible changes in central vision.
  • the present invention features ocular compositions comprising valproic acid and analogs thereof and methods of using such compositions for the treatment or prevention of retinal cell death, including cell death associated with age-related macular degeneration.
  • the invention provides a method for treating or preventing ocular cell death in a subject in need thereof, the method involves administering an effective amount of valproic acid, valpromide, lithium or an analogue thereof to the subject.
  • the invention provides a method for preserving or enhancing visual function, the method involves administering an effective amount of valproic acid, valpromide, lithium or an analogue thereof to the subject.
  • the invention provides a method for increasing retinal regeneration, the method involves administering an effective amount of valproic acid, valpromide, lithium or an analogue thereof to the subject.
  • the ocular cell is a retinal pigment epithelial cell.
  • the ocular cell death is retinal pigment epithelial cell death.
  • the method reduces retinal pigment epithelial cell apoptosis.
  • the method increases the number of retinal pigment epithelial cells.
  • the method increases retinal thickness.
  • the ocular cell death is associated with a disease that is any one or more of retinitis pigmentosa, age-related macular degeneration, glaucoma, corneal dystrophies, retinoschises, Stargardt's disease, autosomal dominant druzen, and Best's macular dystrophy.
  • the disease is the wet or dry form of age-related macular degeneration.
  • the subject contains a mutation that affects opsin folding (e.g., the opsin contains a P23H mutation).
  • the method involves administering valproic acid and lithium or valpromide and lithium.
  • the valproic acid or valpromide and lithium are administered within ten days of each other. In various embodiments of _ any of the above aspects, the valproic acid or valpromide and lithium are administered within one, three, or five days of each other. In various embodiments of any of the above aspects, the valproic acid or valpromide and lithium are administered within twenty-four hours of each other. In various embodiments of any of the above aspects, the valproic acid or valpromide and lithium are administered simultaneously. In various embodiments of any of the above aspects, the valproic acid, valpromide, and/or lithium are administered to the eye (e.g., topical administration).
  • the valproic acid, valpromide, and/or lithium are administered topically by drop form to the surface of the eye. In various embodiments of any of the above aspects, the administration is intra-ocular. In various embodiments of any of the above aspects, the valproic acid, valpromide, and/or lithium are each incorporated into a composition that provides for their long- term release. In various embodiments of any of the above aspects, the method further involves identifying the subject as having or having a propensity to develop ocular cell death, a disease associated with retinal pigmented epithelial cell death, or the wet or dry form of age-related macular degeneration.
  • the invention provides a pharmaceutical composition for treating or preventing ocular cell death in a subject in need thereof, the composition contains an effective amount of valproic acid, valpromide, or lithium and a pharmaceutically acceptable excipient formulated for ocular delivery.
  • the composition is labeled for the treatment of a disease that is any one or more of age-related macular degeneration, retinitis pigmentosa, glaucoma, coreal systrophy, retinoschises, Stargardt's disease, autosomal dominant druzen, or Best's macular dystrophy.
  • the ocular cell is a retinal pigment epithelial cell.
  • the invention features a method for treating or preventing the wet or dry form of age-related macular degeneration in a subject, the method involving administering an effective amount of valproic acid or a derivative thereof to said subject.
  • the invention features a method for treating or preventing retinitis pigmentosa in a subject, the method involving administering an effective amount of valproic acid or a derivative thereof to said subject.
  • the effective amount is between 250 mg/day and 3000 mg/day.
  • the effective amount is 500 mg day, 750 mg/day, 1000 mg/day, 1500 mg day, 2000 mg/day, 2500 mg/day, or 3000 mg day.
  • the method increases best-corrected visual acuity, increases visual field, increases central retinal thickness, or increases subjective visual perception.
  • valproic acid is administered orally, ocularly, topically, or intra- ocularly.
  • the valproic acid is administered topically by drop form to the surface of the eye. In still another embodiment, valproic acid is administered for at least 1 - 12 months, 12-24 months, 36-48 months, or for the life of the patient.
  • the invention provides a kit for the treatment or prevention of ocular cell death, the kit contains an effective amount of valproic acid, valpromide, and/or lithium and instructions for the use of the kit in the method of any of claims 1 - 31. In one embodiment, the kit contains valproic acid and lithium or valpromide and lithium.
  • compositions containing valproic acid and valproic acid analogs and methods of using such compositions to reduce retinal pigment epithelium cell death, particularly cell death associated with age-related macular degeneration.
  • Compositions and articles defined by the invention were isolated or otherwise manufactured in connection with the examples provided below. Other features and advantages of the invention will be apparent from the detailed description, and from the claims.
  • agent any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or fragments thereof.
  • ameliorate decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease.
  • alteration is meant a change (increase or decrease) in the expression levels or activity of a gene or polypeptide as detected by standard art known methods such as those described herein.
  • an alteration includes a 10% change in expression levels, preferably a 25% change, more preferably a 40% change, and most preferably a 50% or greater change in expression levels.
  • analog is meant a molecule that is not identical, but has analogous functional or structural features.
  • a valproic acid analog retains the biological activity of valproic acid, while having certain biochemical modifications that enhance the analog's function relative to unmodified valproic acid.
  • diseases include retinal cell death, such as age-related macular degeneration, retinal detachment, retinal vascular disease, retinitis pigmentosa, glaucoma, diabetic retinopathy, corneal dystrophy, and dry eyes and other diseases associated with retinal pigment epithelium (RPE) cell death.
  • retinal cell death such as age-related macular degeneration, retinal detachment, retinal vascular disease, retinitis pigmentosa, glaucoma, diabetic retinopathy, corneal dystrophy, and dry eyes and other diseases associated with retinal pigment epithelium (RPE) cell death.
  • RPE retinal pigment epithelium
  • an effective amount is meant the amount of a required to ameliorate the symptoms of a disease relative to an untreated patient.
  • the effective amount of active compound(s) used to practice the present invention for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an "effective" amount.
  • increasing cell survival is meant positively altering cell viability.
  • methods that increase cell survival create a corresponding reduction in cell death.
  • Assays for measuring cell viability are known in the art, and are described, for example, by Crouch et al. (J. Immunol. Meth. 160, 81-8); Kangas et al. (Med. Biol.62, 338-43, 1984); Lundin et al., (Meth. Enzymol.133, 27-42, 1986); Petty et al. (Comparison of J. Biolum. Chemilum.10, 29-34, .1995); and Cree et al. (Anticancer Drugs 6: 398 ⁇ 104, 1995).
  • Cell viability can be assayed using a variety of methods, including MTT (3-(4,5-dimethylthiazolyl)-2,5-diphenyltetrazolium bromide)
  • CELLTITER-GLO® Luminescent Cell Viability Assay (Promega), which uses luciferase technology to detect ATP and quantify the health or number of cells in culture, and the CellTiter-Glo® Luminescent Cell Viability Assay, which is a lactate dehyrodgenase (LDH) cytotoxicity assay (Promega).
  • LDH lactate dehyrodgenase
  • cell death is meant necrotic, apoptotic, or any other mechanism resulting in the death of a cell.
  • Assays for measuring cell death are known to the skilled artisan. Apoptotic cells are characterized by characteristic morphological changes, including chromatin condensation, cell shrinkage and membrane blebbing, which can be clearly observed using light microscopy. The biochemical features of apoptosis include DNA fragmentation, protein cleavage at specific locations, increased mitochondrial membrane permeability, and the appearance of phosphatidylserine on the cell membrane surface. Assays for apoptosis are known in the art.
  • Exemplary assays include TUNEL (Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling) assays, caspase activity (specifically caspase-3) assays, and assays for fas-ligand and annexin V.
  • Commercially available products for detecting apoptosis include, for example, Apo-ONE® Homogeneous Caspase-3/7 Assay, FragEL TUNEL kit (ONCOGENE RESEARCH PRODUCTS, San Diego, CA), the ApoBrdU DNA Fragmentation Assay (BIOVISION, Mountain View, CA), and the Quick Apoptotic DNA Ladder Detection Kit (BIOVISION, Mountain View, CA).
  • marker any protein or polynucleotide having an alteration in expression level or activity that is associated with a disease or disorder.
  • obtaining as in “obtaining an agent” includes synthesizing, purchasing, or otherwise acquiring the agent.
  • ocular cell is meant a cell of the eye.
  • An exemplary ocular cell is the retinal pigment epithelial cell.
  • reduces is meant a negative alteration of at least 10%, 25%, 50%, 75%, or 100%.
  • reference is meant a standard or control condition.
  • subject is meant a mammal, including, but not limited to, a human or non-human mammal, such as a bovine, equine, canine, ovine, or feline.
  • Ranges provided herein are understood to be shorthand for all of the values within the range.
  • a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 3j, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, or 50.
  • the terms “treat,” treating,” “treatment,” and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated. Unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms “a”, “an”, and “the” are understood to be singular or plural.
  • the term "about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1 %, 0.5%, 0. 1 %, 0.05%, or 0.01 % of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.
  • compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.
  • Figures 1A-1E are graphs showing that valproic acid (VPA), valpromide (VPD), and (Li treatment reduced hydroquinone (HQ) induced cell death.
  • Figure 1 A is graph showing treatment of ARPE- 19 cells with hydroquinone (HQ) resulted in reduced cell viability in a dose-dependent manner.
  • Figure I B is a graph showing VPA (250 nM to 50 uM) reduced RPE cell death in the MTT assay (B, right panel), when VPA doses were applied concurrently with HQ treatment.
  • Figure 1 C is a graph showing that valproic acid (VPA) protects ARPE- 19 cells against cell death induced by 250 ⁇ and 350 ⁇ hydroquinone treatment.
  • VPD denotes valpromide and Li denotes lithium.
  • Figure ID is a graph showing that VPA protects RPE cells in a dose dependent manner against HQ mediated cell death.
  • Figure IE is a graph showing that VPD was not protective of RPE cell death at a range of ⁇ - 50 ⁇ (v. protective effect at 250 ⁇ in Figure 1 C).
  • Figure 2 is a graph showing that valproic acid (VPA) prevented hydroquinone- induced apoptosis in ARPE- 19 cells treated with 250 ⁇ and 350 ⁇ hydroquinone.
  • Figure 3 is a graph showing Tunel staining in ARPE-19 cells treated with valproic acid (VPA), valpromide (VPD) and/or Lithium.
  • Figures 4A-4D depict scotopic ERG and histology results from P23H mutant mice treated with VPA and vehicle.
  • Figure 4B is a graph showing scotopic ERG b-wave amplitude from mice treated for 1 1 weeks
  • Figure 4C depicts a cross-section of retina from a control mouse.
  • Figures 5A and 5B are box and whiskers diagrams showing increases in central retinal thickness and photopic ERG b-wave amplitude from mer tk-/- mice treated with either with VPA (250 mg/kg).
  • Figure 5A is a box and whiskers diagram depicting results for central retinal thickness as determined by ultra-high resolution OCT and measured at -500 microns from the center of the optic disc either temporally (abbreviated “temp") or nasally (abbreviated "nas”) at the end of a 4 week daily i.p. treatment with either VPA or vehicle (control group).
  • Figure 5B is a box and whiskers diagram depicting amplitude of the photopic b-wave measured at the same time from the two groups.
  • Figures 6A-6C are box and whiskers diagrams showing increases in central retinal thickness and photopic ERG b-wave amplitude after sodium iodate injection (20mg/kg; i.v.) in C57BL/6 mice treated with VPA.
  • Figures 6A and 6B are box and whiskers diagrams depicting results for central retinal thickness as determined by ultra-high resolution OCT and measured at -500 microns from the center of the optic disc either temporally (abbreviated “temp") or nasally (abbreviated “nas”) at the end of Day 9 ( Figure 6A) and Day 15 ( Figure 6B) after sodium iodate injection for mice treated with either VPA or vehicle (control group).
  • Figure 6C is a box and whiskers diagram depicting the amplitude of the photopic b-wave measured at Day 10 from the two groups mice of mice treated with VPA or vehicle (control group). Statistical significance of a comparison between the VPA-treated and control group (Mann- Whitney U test) is shown for each comparison pair.
  • Figures 7A-7D are box and whiskers diagrams ( Figures 7A, 7C, 7D) or graphs ( Figure 7B) showing a change in BCVA at follow-up visit compared to baseline visit
  • Figure 7A shows BCVA results averaged for left eye and right eye: only patient data for both eyes was used; also expected BCVA value from the natural history of the disease is shown as a comparison.
  • Figure 7B indicates the number of eyes showing improvement, no change, or worsening of BCVA at follow-up, by daily treatment dose of VPA.
  • Figure 7C shows BCVA change as a function of daily dose and provides a comparison of observed results to the value expected based on the natural history of the disease; only patient data for both eyes was used.
  • Figure 7D presents the same results as Figure 7C, but instead of an average of both eyes, only the results for the more improved eye were used. .
  • Figures 8A and 8B are box and whiskers diagrams showing the change in BCVA at a follow-up visit compared to a baseline visit for eyes with dry ARMD treated with 500 mg or 750 mg VPA per day, and compared to the value expected from the natural history of the disease.
  • Figure 8A shows one result per patient included; in cases where two eyes had the same diagnosis, the result was averaged.
  • Figure 8B presents the same results as Figure 8A, but when data for two eyes were available, only the result from the more improved eye was included.
  • Figures 9A-9C show treatment results for eyes with wet ARMD.
  • Figure 9A shows the change in BCVA at a follow-up visit compared to a baseline visit for wet AMD patients treated with 500 mg or 750 mg VPA per day. One result per patient was included; in cases where two eyes had the same diagnosis, the results were averaged. The value expected from the natural history of the disease is also shown for comparison (Wong et al. 2007).
  • Figure 9B shows the results for eyes with decreased or increased central retinal thickness as measured by OCT.
  • Figure 9C shows a representative OCT from the left eye of patient #21 at baseline (upper panel) and at follow-up (lower panel).
  • FIGS 10A and 10B show the effect on visual field in patients treated with
  • Figure 10A provides Goldmann Kinetic Perimetry tracings from patient 6 at baseline (left) and after 6 months of VPA treatment (middle -red), overlap of baseline and follow-up (right).
  • Figure 10B quantitates the observed change in Visual Field: Baseline and follow-up VF areas are graphed for each individual patient, length of follow-up varied for each patient. Areas were analyzed by right eye, left eye and average of both eyes. Numbers correspond to the individual subjects.
  • Figure 1 1 shows an analysis of change in visual field in patients treated with VPA.
  • Goldmann Kinetic Perimetry tracings (isopter V4e) from each eye were digitized and areas (mm2) were calculated as described in methods and log transformed. Scatterplots of change in log transformed VF over the course of treatment are shown for left eyes, right eyes and all eyes combined. Mean value is shown by thin bar and standard deviation is represented by upper and lower dark bars.
  • the invention features compositions and methods that are useful for the treatment or prevention of ocular cell death.
  • the invention is based, at least in part on the discovery that valproic acid reduced cell death both in vitro and in vivo models of retinal pigment epithelium (RPE) damage.
  • RPE retinal pigment epithelium
  • valproic acid increased visual function in patients with retinitis pigmentosa and age-related macular degeneration.
  • ARPE- 19 cell death was induced by contacting the cells in vitro with 200-350 ⁇ HQ. More than 60% of cells treated with 250 ⁇ HQ or more died.
  • VPA treatment (500nM - 250 ⁇ ) significantly increased the number of viable cells at 48 hours. Annexin V staining showed that the number of apoptotic cells increased as the hydroquinone dosage increased.
  • VPA retinal pigment epithelium
  • ARMD which is the leading cause of blindness in people over 60 in the developed world, is characterized by loss of central visual function, including visual acuity. ARMD is categorized into two major forms: an angiogenic, wet form and a non-exudative, dry form. Recent emerging technologies and novel therapeutics have inadequately targeted the physiological aspects and progression of wet ARMD, which has a faster disease progression and poorer visual prognosis than dry ARMD. Treatment options have emerged to suppress choroidal neovascularization, the hallmark of wet ARMD, such as photocoagulation, photodynamic therapy, and anti- VEGF therapies, including monoclonal antibodies and corticosteroids. These treatments may temporarily stabilize and improve visual function in wet ARMD.
  • AREDS Age-Related Eye Disease Study
  • ARMD is a growing public health concern with an increasingly older population.
  • Wet ARMD accounts for 10%-20% of ARMD patients and accounts for 90% of those with severe vision loss. Dry ARMD can also lead to significant vision loss.
  • the fundamental characteristics of ARMD are an inflammatory reaction of the RPE and photoreceptor cells and subsequent cell death. Contributing to this process are photo- oxidative stress, complement activation, immune dysregulation, and inflammatory cell infiltration.
  • none of the currently available therapies address the underlying pathophysiology of dry or wet ARMD.
  • valproic acid is a safe and effective therapy for non-exudative ("dry") or exudative ("wet") ARMD in humans that exerts its biological effect in the retina by protecting photoreceptor cells, reducing inflammatory pro-angiogenic cytokines in the RPE, limiting oxidative apoptotic injury and mitigating alternative complement pathway-activated cell death.
  • VPA and analogs and derivatives thereof have a biological profile that is well suited for treatment of retinal diseases.
  • human studies in patients with ARMD have a biological profile that is well suited for treatment of retinal diseases.
  • Retinitis pigmentosa Retinitis pigmentosa is a severe neurodegenerative disease of the retina characterized initially by night blindness, with progression to tunnel vision and eventual loss of central vision and total blindness.
  • Targeted therapies for RP are complicated by the identification of more than 40 genes linked to the dominant and recessive forms of this disease. While a few new approaches for RP treatment have recently been investigated, including nutritional supplementation, light reduction, and gene therapy, of these, vitamin A supplementation is the most promising, but its benefits are modest and side effects are problematic. Therefore, currently there is no significant treatment or cure for RP.
  • VPA is a potent inhibitor of histone deacetylase (HDAC) (Gottlich et al., Valproic acid defines a novel class of HDAC inhibitors inducing differentiation of transformed cells. EMBO J. 2001 ;20:6969-78) and the inflammatory response pathway via apoptosis of microglial cells (Dragunow et al., Neuroscience. 2006; 140: 1 149-56; Chen et al., Neuroscience. 2007; 149:203-12; Kim et al., J Pharmacol Exp Ther. 2007;321 :892-901). In addition, VPA down-regulates complement proteins (Suuronen et al., Biochem Biophys Res Commun.
  • HDAC histone deacetylase
  • VPA has a unique biological profile suitable for treating retinal diseases.
  • VPA and its derivative, divalproex sodium have also been used for chronic pain syndromes, cancer therapy and schizophrenia.
  • VPA is also useful for the treatment of patients with retinal dystrophies as shown in an analysis of the efficacy of VPA treatment on vision function of patients with RP.
  • Valproic acid is a histone deacetylate inhibitor.
  • the invention provides for the use of valproic acid, valpromide, and analogs thereof to prevent or reduce RPE cell death in a subject at risk thereof (e.g., a subject diagnosed as having or having a propensity to develop age-related macular degeneration).
  • Valproic acid, valpromide, and analogs thereof may be administered alone or in combination with lithium to ameliorate RPE cell death, particularly cell death associated with the wet or dry form of age-related macular degeneration.
  • a pharmaceutical composition includes valproic acid or valpromide in combination with lithium.
  • the valproic acid or valpromide and the lithium are formulated together or separately.
  • Compounds of the invention may be administered as part of a pharmaceutical composition.
  • the compositions should be sterile and contain a therapeutically effective amount of the polypeptides in a unit of weight or volume suitable for administration to a subject.
  • the compositions and combinations of the invention can be part of a pharmaceutical pack, where each of the compounds is present in individual dosage amounts.
  • compositions of the invention to be used for prophylactic or therapeutic administration should be sterile. Sterility is readily accomplished by filtration through sterile filtration membranes (e.g., 0.2 ⁇ membranes), by gamma irradiation, or any other suitable means known to those skilled in the art.
  • Therapeutic polypeptide compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
  • These compositions ordinarily will be stored in unit or multi-dose containers, for example, sealed ampoules or vials, as an aqueous solution or as a lyophilized formulation for reconstitution.
  • the compounds may be combined, optionally, with a pharmaceutically acceptable excipient.
  • pharmaceutically-acceptable excipient means one or more compatible solid or liquid filler, diluents or encapsulating substances that are suitable for administration into a human.
  • carrier denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate administration.
  • the components of the pharmaceutical compositions also are capable of being co-mingled with the molecules of the present invention, and with each other, in a manner such that there is no interaction that would substantially impair the desired pharmaceutical efficacy.
  • Compounds of the present invention can be contained in a pharmaceutically acceptable excipient.
  • the excipient preferably contains minor amounts of additives such as substances that enhance isotonicity and chemical stability.
  • Such materials are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, succinate, acetate, lactate, tartrate, and other organic acids or their salts; tris- hydroxymethylaminomethane (TRIS), bicarbonate, carbonate, and other organic bases and their salts; antioxidants, such as ascorbic acid; low molecular weight (for example, less than about ten residues) polypeptides, e.g., polyarginine, polylysine, polyglutamate and polyaspartate; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as
  • polyvinylpyrrolidone PVP
  • polypropylene glycols PPGs
  • polyethylene glycols PEGs
  • amino acids such as glycine, glutamic acid, aspartic acid, histidine, lysine, or arginine
  • monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, mannose, sucrose, dextrins or sulfated carbohydrate derivatives, such as heparin, chondroitin sulfate or dextran sulfate
  • polyvalent metal ions such as divalent metal ions including calcium ions, magnesium ions and manganese ions
  • chelating agents such as ethylenediamine tetraacetic acid (EDTA)
  • sugar alcohols such as mannitol or sorbitol
  • counterions such as sodium or ammonium
  • nonionic surfactants such as polysorbates or poloxamers.
  • additives such as stabilizers, anti-microbials, inert gases, fluid and nutrient replenishers (i.e., Ringer's dextrose), electrolyte replenishers, and the like, which can be present in conventional amounts.
  • compositions as described above, can be administered in effective amounts.
  • the effective amount will depend upon the mode of administration, the particular condition being treated and the desired outcome. It may also depend upon the stage of the condition, the age and physical condition of the subject, the nature of concurrent therapy, if any, and like factors well known to the medical practitioner. For therapeutic applications, it is that amount sufficient to achieve a medically desirable result.
  • an effective amount is sufficient to reduce cell death, increase cell viability, and/or increase the level of a correctly folded protein in a cell.
  • an effective amount is an amount sufficient to stabilize, slow, or reduce the a symptom associated with a pathology.
  • doses of the compounds of the present invention would be from about 0.01 mg/kg per day to about 1000 mg/kg per day. It is expected that doses ranging from about 50 to about 2000 mg/kg will be suitable. Lower doses will result from certain forms of administration, such as intravenous administration.
  • a composition of the invention is administered intraocularly.
  • Other modes of administration include oral, topical, intraocular, buccal, transdermal, within/on implants, or parenteral routes.
  • parenteral includes
  • compositions comprising a composition of the invention can be added to a physiological fluid, such as to the intravitreal humor.
  • compositions of the invention can comprise one or more pH buffering compounds to maintain the pH of the formulation at a predetermined level that reflects physiological pH, such as in the range of about 5.0 to about 8.0.
  • the pH buffering compound used in the aqueous liquid formulation can be an amino acid or mixture of amino acids, such as histidine or a mixture of amino acids such as histidine and glycine.
  • the pH buffering compound is preferably an agent which maintains the pH of the formulation at a predetermined level, such as in the range of about 5.0 to about 8.0, and which does not chelate calcium ions.
  • Illustrative examples of such pH buffering compounds include, but are not limited to, imidazole and acetate ions.
  • compositions of the invention can also contain one or more osmotic modulating agents, i.e., a compound that modulates the osmotic properties (e.g, tonicity, osmolality and/or osmotic pressure) of the formulation to a level that is acceptable to the blood stream and blood cells of recipient individuals.
  • osmotic modulating agent can be an agent that does not chelate calcium ions.
  • the osmotic modulating agent can be any compound known or available to those skilled in the art that modulates the osmotic properties of the formulation.
  • osmotic modulating agents include, but are not limited to: salts, such as sodium chloride and sodium acetate; sugars, such as sucrose, dextrose, and mannitol; amino acids, such as glycine; and mixtures of one or more of these agents and/or types of agents.
  • the osmotic modulating agent(s) may be present in any concentration sufficient to modulate the osmotic properties of the formulation.
  • compositions comprising a compound of the present invention can contain multivalent metal ions, such as calcium ions, magnesium ions and/or manganese ions. Any multivalent metal ion that helps stabilizes the composition and that will not adversely affect recipient individuals may be used. The skilled artisan, based on these two criteria, can determine suitable metal ions empirically and suitable sources of such metal ions are known, and include inorganic and organic salts.
  • compositions of the invention can also be a non-aqueous liquid formulation.
  • Any suitable non-aqueous liquid may be employed, provided that it provides stability to the active agents (s) contained therein.
  • the nonaqueous liquid is a hydrophilic liquid.
  • suitable non-aqueous liquids include: glycerol; dimethyl sulfoxide (DMSO); polydimethylsiloxane (PMS); ethylene glycols, such as ethylene glycol, diethylene glycol, triethylene glycol, polyethylene glycol ("PEG”) 200, PEG 300, and PEG 400; and propylene glycols, such as dipropylene glycol, tripropylene glycol, polypropylene glycol ("PPG”) 425, PPG 725, PPG 1000, PPG 2000, PPG 3000 and PPG 4000.
  • DMSO dimethyl sulfoxide
  • PMS polydimethylsiloxane
  • ethylene glycols such as ethylene glycol, diethylene glycol, triethylene glycol, polyethylene glycol (“PEG”) 200, PEG 300, and PEG 400
  • PEG polyethylene glycol
  • PPG polypropylene glycol
  • PPG polypropylene glycol
  • compositions of the invention can also be a mixed
  • aqueous/non-aqueous liquid formulation aqueous/non-aqueous liquid formulation.
  • Any suitable non-aqueous liquid formulation such as those described above, can be employed along with any aqueous liquid formulation, such as those described above, provided that the mixed aqueous/non-aqueous liquid formulation provides stability to the compound contained therein.
  • the non- aqueous liquid in such a formulation is a hydrophilic liquid.
  • suitable non-aqueous liquids include: glycerol;
  • DMSO methyl methacrylate
  • PMS ethylene glycols
  • ethylene glycols such as PEG 200, PEG 300, and PEG 400
  • propylene glycols such as PPG 425, PPG 725, PPG 1000, PPG 2000, PPG 3000 and PPG 4000.
  • Suitable stable formulations can permit storage of the active agents in a frozen or an unfrozen liquid state.
  • Stable liquid formulations can be stored at a temperature of at least -70°C, but can also be stored at higher temperatures of at least 0°C, or between about 0.1 °C and about 42°C, depending on the properties of the composition. It is generally known to the skilled artisan that proteins and polypeptides are sensitive to changes in pH, temperature, and a multiplicity of other factors that may affect therapeutic efficacy.
  • a desirable route of administration can be by pulmonary aerosol.
  • Techniques for preparing aerosol delivery systems containing polypeptides are well known to those of skill in the art. Generally, such systems should utilize components that will not significantly impair the biological properties of the antibodies, such as the paratope binding capacity (see, for example, Sciarra and Cutie, "Aerosols," in Remington's Pharmaceutical Sciences, 18th edition, 1990, pp 1694- 1712; incorporated by reference). Those of skill in the art can readily modify the various parameters and conditions for producing polypeptide aerosols without resorting to undue experimentation.
  • Other delivery systems can include time-release, delayed release or sustained release delivery systems. Such systems can avoid repeated administrations of compositions of the invention, increasing convenience to the subject and the physician.
  • Many types of release delivery systems are available and known to those of ordinary skill in the art. They include polymer base systems such as polylactides (U.S. Pat. No. 3,773,919; European Patent No. 58,481 ), poly(lactide-glycolide), copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters,
  • polyhydroxybutyric acids such as poly-D-(-)-3-hydroxybutyric acid (European Patent No. 133, 988), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate
  • sustained-release compositions include semi-permeable polymer matrices in the form of shaped articles, e.g., films, or microcapsules.
  • Delivery systems also include non-polymer systems that are: lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono- di- and tri-glycerides; hydrogel release systems such as biologically-derived lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono- di- and tri-glycerides; hydrogel release systems such as biologically-derived lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono- di- and tri-glycerides; hydrogel release systems such as biologically-derived
  • bioresorbable hydrogel i.e., chitin hydrogels or chitosan hydrogels
  • sylastic systems i.e., chitin hydrogels or chitosan hydrogels
  • peptide based systems e.g., a-(2-aminoethyl)
  • wax coatings e.g., a-(2-aminoethyl)
  • compressed tablets using conventional binders and excipients e.g., partially fused implants
  • partially fused implants i.e., chitin hydrogels or chitosan hydrogels
  • Specific examples include, but are not limited to: (a) erosional systems in which the agent is contained in a form within a matrix such as those described in U.S. Patent Nos. 4,452,775, 4,667,014, 4,748,034 and 5,239,660 and (b) diffusional systems in which an active component permeates at a controlled rate from a polymer such
  • colloidal dispersion systems include lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • Liposomes are artificial membrane vessels, which are useful as a delivery vector in vivo or in vitro.
  • Large unilamellar vessels (LUV) which range in size from 0.2 - 4.0 ⁇ , can encapsulate large macromolecules within the aqueous interior and be delivered to cells in a biologically active form (Fraley, R., and Papahadjopoulos, D., Trends Biochem. Sci. 6: 77-80).
  • Liposomes can be targeted to a particular tissue by coupling the liposome to a specific ligand such as a monoclonal antibody, sugar, glycolipid, or protein.
  • a specific ligand such as a monoclonal antibody, sugar, glycolipid, or protein.
  • Liposomes are commercially available from Gibco BRL, for example, as
  • LIPOFECTINTM and LIPOFECTACETM which are formed of cationic lipids such as N-[ l -(2, 3 dioleyloxy)-propyl]-N, N, N-trimethylammonium chloride (DOTMA) and dimethyl dioctadecylammonium bromide (DDAB).
  • DOTMA N-[ l -(2, 3 dioleyloxy)-propyl]-N, N, N-trimethylammonium chloride
  • DDAB dimethyl dioctadecylammonium bromide
  • PCT/US/03307 Publication No. WO 95/24929, entitled “Polymeric Gene Delivery System”
  • PCT/US/0307 describes biocompatible, preferably biodegradable polymeric matrices for containing an exogenous gene under the control of an appropriate promoter.
  • the polymeric matrices can be used to achieve sustained release of the exogenous gene or gene product in the subject.
  • the polymeric matrix preferably is in the form of a microparticle such as a microsphere (wherein an agent is dispersed throughout a solid polymeric matrix) or a microcapsule (wherein an agent is stored in the core of a polymeric shell).
  • a microparticle such as a microsphere (wherein an agent is dispersed throughout a solid polymeric matrix) or a microcapsule (wherein an agent is stored in the core of a polymeric shell).
  • Microcapsules of the foregoing polymers containing drugs are described in, for example, U.S. Patent 5,075, 109.
  • Other forms of the polymeric matrix for containing an agent include films, coatings, gels, implants, and stents.
  • the size and composition of the polymeric matrix device is selected to result in favorable release kinetics in the tissue into which the matrix is introduced.
  • the size of the polymeric matrix further is selected according to the method of delivery that is to be used.
  • the polymeric matrix and composition are encompassed in a surfactant vehicle.
  • the polymeric matrix composition can be selected to have both favorable degradation rates and also to be formed of a material, which is a bioadhesive, to further increase the effectiveness of transfer.
  • the matrix composition also can be selected not to degrade, but rather to release by diffusion over an extended period of time.
  • the delivery system can also be a biocompatible microsphere that is suitable for local, site-specific delivery. Such microspheres are disclosed in
  • Both non-biodegradable and biodegradable polymeric matrices can be used to deliver the compositions of the invention to the subject.
  • Such polymers may be natural or synthetic polymers.
  • the polymer is selected based on the period of time over which release is desired, generally in the order of a few hours to a year or longer. Typically, release over a period ranging from between a few hours and three to twelve months is most desirable.
  • the polymer optionally is in the form of a hydrogel that can absorb up to about 90% of its weight in water and further, optionally is cross- linked with multivalent ions or other polymers.
  • Exemplary synthetic polymers which can be used to form the biodegradable delivery system include: polyamides, polycarbonates, polyalkylenes, polyalkylene glycols, polyalkylene oxides, polyalkylene terepthalates, polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, poly-vinyl halides, polyvinylpyrrolidone, polyglycolides, polysiloxanes, polyurethanes and co-polymers thereof, alkyl cellulose, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, polymers of acrylic and methacrylic esters, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxy-propyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxylethyl cellulose, cellulose tri
  • compositions of the invention are particularly suitable for treating diseases characterized by retinal cell death, such as age-related macular degeneration, retinal detachment, retinal vascular disease, retinitis pigmentosa, glaucoma, diabetic retinopathy, corneal dystrophy, and dry eyes.
  • diseases characterized by retinal cell death such as age-related macular degeneration, retinal detachment, retinal vascular disease, retinitis pigmentosa, glaucoma, diabetic retinopathy, corneal dystrophy, and dry eyes.
  • compositions of the invention are administered through an ocular device suitable for direct implantation into the vitreous of the eye.
  • the compositions of the invention may be provided in sustained release compositions, such as those described in, for example, U.S. Pat. Nos. 5,672,659 and 5,595,760. Such devices are found to provide sustained controlled release of various agents.
  • compositions to treat the eye without risk of detrimental local and systemic side effects are provided.
  • An object of the present ocular method of delivery is to maximize the amount of drug contained in an intraocular device or implant while minimizing its size in order to prolong the duration of the implant. See, e.g., U.S. Patents 5,378,475;
  • Such implants may be biodegradable and/or biocompatible implants, or may be non- biodegradable implants.
  • Biodegradable ocular implants are described, for example, in U.S. Patent Publication No. 20050048099.
  • the implants may be permeable or impermeable to the active agent, and may be inserted into a chamber of the eye, such as the anterior or posterior chambers or may be implanted in the schlera,
  • transchoroidal space or an avascularized region exterior to the vitreous.
  • a contact lens that acts as a depot for compositions of the invention may also be used for drug delivery.
  • the implant may be positioned over an avascular region, such as on the sclera, so as to allow for transcleral diffusion of the drug to the desired site of treatment, e.g. the intraocular space and macula of the eye.
  • the site of transcleral diffusion is preferably in proximity to the macula.
  • implants for delivery of an a composition include, but are not limited to, the devices described in U.S. Pat. Nos. 3,416,530; 3,828,777; 4,014,335; 4,300,557; 4,327,725; 4,853,224; 4,946,450; 4,997,652; 5, 147,647; 5,164, 188; 5,178,635;
  • a sustained release drug delivery system comprising an inner reservoir comprising an effective amount of an agent effective in obtaining a desired local or systemic physiological or pharmacological effect, an inner tube impermeable to the passage of the agent, the inner tube having first and second ends and covering at least a portion of the inner reservoir, the inner tube sized and formed of a material so that the inner tube is capable of supporting its own weight, an impermeable member positioned at the inner tube first end, the impermeable member preventing passage of the agent out of the reservoir through the inner tube first end, and a permeable member positioned at the inner tube second end, the permeable member allowing diffusion of the agent out of the reservoir through the inner tube second end; a method for administering a compound of the invention to a segment of an eye, the method comprising the step of implanting a sustained release device to deliver the compound of the invention to the vitreous of the eye or an implantable, sustained release device for administering a compound of the invention to a segment of
  • liposomes to target a compound of the present invention to the eye, and preferably to retinal pigment epithelial cells and/or Bruch's membrane.
  • the compound may be complexed with liposomes in the manner described above, and this
  • the compound/liposome complex injected into patients with an ocular PCD, using intravenous injection to direct the compound to the desired ocular tissue or cell.
  • Directly injecting the liposome complex into the proximity of the retinal pigment epithelial cells or Bruch's membrane can also provide for targeting of the complex with some forms of ocular PCD.
  • the compound is administered via intra-ocular sustained delivery (such as VITRASERT or
  • the compound is delivered by posterior subtenons injection.
  • microemulsion particles containing the compositions of the invention are delivered to ocular tissue to take up lipid from Bruch's membrane, retinal pigment epithelial cells, or both.
  • Nanoparticles are a colloidal carrier system that has been shown to improve the efficacy of the encapsulated drug by prolonging the serum half-life.
  • Polyalkylcyanoacrylates (PACAs) nanoparticles are a polymer colloidal drug delivery system that is in clinical development, as described by Stella et al., J. Pharm. Sci., 2000. 89: p. 1452- 1464; Brigger et al., Int. J. Pharm., 2001. 214: p. 37-42; Calvo et al., Pharm. Res., 2001. 18: p. 1 157-1 166; and Li et al., Biol. Pharm. Bull., 2001. 24: p. 662-665.
  • Biodegradable poly (hydroxyl acids) such as the copolymers of poly (lactic acid) (PLA) and poly (lactic-co-glycolide) (PLGA) are being extensively used in biomedical applications and have received FDA approval for certain clinical applications.
  • PEG-PLGA nanoparticles have many desirable carrier features including (i) that the agent to be encapsulated comprises a reasonably high weight fraction (loading) of the total carrier system; (ii) that the amount of agent used in the first step of the encapsulation process is incorporated into the final carrier
  • Nanoparticles are synthesized using virtually any biodegradable shell known in the art.
  • a polymer such as poly (lactic-acid) (PLA) or poly (lactic-co-glycolic acid) (PLGA) is used.
  • PLA poly (lactic-acid)
  • PLGA poly (lactic-co-glycolic acid)
  • Such polymers are biocompatible and biodegradable, and are subject to modifications that desirably increase the
  • the polymer is modified with a terminal carboxylic acid group (COOH) that increases the negative charge of the particle and thus limits the interaction with negatively charge nucleic acid aptamers.
  • COOH carboxylic acid group
  • Nanoparticles are also modified with polyethylene glycol (PEG), which also increases the half-life and stability of the particles in circulation.
  • PEG polyethylene glycol
  • the COOH group is converted to an N- hydroxysuccinimide (NHS) ester for covalent conjugation to amine-modified aptamers.
  • Biocompatible polymers useful in the composition and methods of the invention include, but are not limited to, polyamides, polycarbonates, polyalkylenes, polyalkylene glycols, polyalkylene oxides, polyalkylene terepthalates, polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, polyvinyl halides, polyvinylpyrrolidone, polyglycolides, polysiloxanes, polyurethanes and copolymers thereof, alkyl cellulose, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, polymers of acrylic and methacrylic esters, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxy-propyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetage phthalate, carboxylethyl cellulose, cellulose tria
  • the nanoparticles of the invention include PEG-PLGA polymers.
  • compositions of the invention may also be delivered topically.
  • the compositions are provided in any pharmaceutically acceptable excipient that is approved for ocular delivery.
  • the composition is delivered in drop form to the surface of the eye.
  • the delivery of the composition relies on the diffusion of the compounds through the cornea to the interior of the eye.
  • Human dosage amounts can initially be determined by extrapolating from the amount of compound used in mice, as a skilled artisan recognizes it is routine in the art to modify the dosage for humans compared to animal models.
  • the dosage may vary from between about 1 mg compound/Kg body weight to about 5000 mg compound/Kg body weight; or from about 5 mg/Kg body weight to about 4000 mg/Kg body weight or from about 10 mg/Kg body weight to about 3000 mg/Kg body weight; or from about 50 mg/Kg body weight to about 2000 mg/Kg body weight; or from about 100 mg/Kg body weight to about 1000 mg/Kg body weight; or from about 150 mg/Kg body weight to about 500 mg/Kg body weight.
  • this dose may be about 1 , 5, 10, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1 100, 1 150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, 3500, 4000, 4500, 5000 mg/Kg body weight. In other embodiments, it is envisaged that higher does may be used, such doses may be in the range of about 5 mg compound/Kg body to about 20 mg compound/Kg body.
  • the doses may be about 8, 10, 12, 14, 16 or 18 mg/Kg body weight.
  • this dosage amount may be adjusted upward or downward, as is routinely done in such treatment protocols, depending on the results of the initial clinical trials and the needs of a particular patient.
  • kits for the treatment or prevention of RPE cell death includes a pharmaceutical pack comprising an effective amount of valproic acid, valpromide, lithium, or combinations thereof.
  • the compositions are present in unit dosage form.
  • the kit comprises a sterile container which contains a therapeutic or prophylactic
  • compositions such containers can be boxes, ampoules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art.
  • Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.
  • compositions of the invention or combinations thereof are provided together with instructions for administering them to a subject having or at risk of developing RPE cell death.
  • the instructions will generally include information about the use of the compounds for the treatment or prevention of RPE cell death.
  • the instructions include at least one of the following: description of the compound or combination of compounds; dosage schedule and administration for treatment of a disease characterized by RPE cell death (e.g., age-related macular degeneration) or symptoms thereof; precautions; warnings; indications; counter- indications; overdosage information; adverse reactions; animal pharmacology; clinical studies; and/or references.
  • the instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.
  • Example 1 VpA, Valpromide (VpD), and Li treatment ameliorated HQ induced cell death
  • HQ hydroquinone
  • MTT conversion of 3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) to a purple dye is an indicator of live cells.
  • FIG. 1 A A cell viability assay was performed with a variety of HQ concentrations (Figure 1 A). Significant cell death was observed at 250 ⁇ HQ with cell mortality rapidly increasing at successively higher concentrations. Incubation with 900hM VpA, 250 ⁇ VpD, and 4mM Li lead to a significant reduction in cell death with an even greater increase in the Li and VpA combination treatment at 250 ⁇ HQ ( Figure 1C). At 350 ⁇ HQ, all four interventions significantly increased cell survival. 250 ⁇ valpromide (VpD), and the combination of Li + VpA treatment showed slightly greater cell survival that valproic acid alone. At 450 ⁇ HQ, none of the treatments showed any cytoprotective effects.
  • VPA protected RPE cells in a dose dependent manner against HQ mediated cell death with a significant increase in cell viability at 250nM VPA (Figure I D).
  • VPD was not protective of RPE cell death at a range of 10 ⁇ -50 ⁇ in the oxidative stress model of hydroquinone (HQ)-induced RPE cell death (Figure I E), although a protective effect was observed at 250 ⁇ VPD ( Figure 1 C).
  • HQ hydroquinone
  • VPD valproic acid
  • VPD valpromide
  • HQ Li treatment reduced hydroquinone
  • Example 2 VpA, VpD, and Li treatment ameliorate HQ induce apoptosis
  • interventions significantly reduce the annexin V positive (early apoptotic) fraction and reduce the overall amount of apoptosis. When incubated with 450 ⁇ HQ, the interventions show no anti-apoptotic effects.
  • VpA pre-treatment exerted no anti- apoptotic activity at any concentration of HQ.
  • the VpA treated cells displayed a similar apoptotic profile to the HQ treated cells in HSFl deficient MEFs.
  • Example 4 VpA, VpD, and Li treatment exhibited multipotent anti-apoptogenic effects
  • TUNEL staining was performed (Figure 3). At 250 ⁇ HQ, 900nM Vpa, 2mM Li, and 250 ⁇ VpD reduced the TUNEL stain intensity by about half. At 350 ⁇ HQ, the same three interventions significantly reduced the level of TUNEL staining. At 450 ⁇ HQ, there were no significant anti- apoptotic effects observed in response to VpA, VpD, or Li pre-treatment.
  • Example 5 VPA reduces cytokine levels in RPE cell monolayer.
  • cytokine levels were assayed at fixed time points from apical and basolateral surfaces in RPE cultures.
  • Treatment with IL- ⁇ ⁇ , IFN- ⁇ , TNF-a (ICM) was used to induce a cytokine response.
  • VPA was tested at 10 ⁇ .
  • Sandwich Enzyme-linked immunosorbent assay (ELISA) was performed to detect cytokine levels using a commercially available sensitive, fluorescent detection system (Searchlight).
  • Example 6 VPA increases retinal formation in mouse models of retinal disease or retinal damage.
  • P23H transgenic mice (line 37) contain a human P23H RHO transgene including the entire rhodopsin gene transcriptional unit plus 4.2 kb of upstream and 8.4 kb of downstream DNA.
  • Founder P23H rhodopsin mice (on an FVB background) were backcrossed with C57BL/6J mice for 10 generations to obtain human transgenic mice on a uniform B6 genetic background. This line contains an equal number of copies of the human rhodopsin transgene and the endogenous mouse rhodopsin gene.
  • mice Male and female P23H mice were treated for 1 1 - 12 weeks (with a subgroup treated for 20 weeks) with an i.p. injection of VPA (250 mg/kg) or vehicle once every two days starting from age P25-P30.
  • VPA 250 mg/kg
  • Full-field ERGs were recorded at baseline and after 1 1 weeks of treatment. Vision function was evaluated by ERG recordings (Espion E2 with ColorDome, Diagnosys LLC, Lowell, MA). Mice were dark-adapted overnight. Subdermal needle electrodes served as reference and ground, while a contact lens electrode served as active.
  • Full-field ERG recordings were obtained from both eyes from white flashes of increasing intensities (0.02, 0.26, 2.8, 28 and 100 cd.s/m2) and b-waves of the recorded traces were measured and analyzed.
  • the bandpass filter was set between 0.3 and 300 Hz.
  • the averaged responses were measured in a conventional way (from the trough of the a-wave) to obtain b-wave amplitude.
  • mice were anesthetized (ketamine 100 mg/kg and xylaizine 10 m kg mixture) and pupils dilated with 1 drop of phenylephrine hydrochloride 2.5% and tropicamide 1 %.
  • Full retinal thickness at -500 microns nasally and temporally from the optic disc was measured with ultrahigh resolution OCT (Bioptigen Inc., Durham, NC). The nasal and temporal measurements from both eyes were averaged across the eyes and analyzed separately.
  • mice were anesthetized and pupil dilated in a similar manner as for OCT recording. Vision function was evaluated by full-field ERG recordings (Espion E2 with ColorDome, Diagnosys LLC, Lowell, MA). Specifically, after 9 minutes of light adaptation, photopic ERG traces were obtained from both eyes as a result of a white flash stimulation with an intensity of 10 phot cd.s/m2 , at l Hz frequency presented on a 34 cd/m2 white background. The bandpass filter was set between 0.3 and 300 Hz.
  • Sodium iodate induced retinal damage in mice is a model of Age related macular degeneration (ARMD).
  • the RPE is the initial site of the toxic action of sodium iodate, with secondary effects exerted on photoreceptor changes.
  • ERG response disappears by Day 3 after dosing at 40 mg/kg, however the response is preserved at -50% level and stays relatively stable for a couple of weeks after dosing with 20 mg/kg.
  • the process of ongoing retinal apoptosis is most pronounced at Day 3 and the intensity of the process rapidly declines, with very little apoptosis present at Day 28. (Machalinska et al., 2010).
  • Example 7 VPA provides an effective treatment for human subjections with ARMD
  • ARMD age-related macular degeneration
  • VPA valproic acid
  • VPA should be considered as a treatment for both wet and dry ARMD.
  • VPA had minimal effect on BCVA and retinal thickness.
  • VPA was associated with a subjective improvement in visual functioning and a statistically significant improvement in BCVA.
  • VPA provides a promising new treatment for the dry and wet forms of ARMD consistent with current understanding of the pathophysiology of the disease and the known biological properties of existing therapies.
  • a single patient reported a side effect (severe fatigue) that was not transient. Without exception, all transient side effects subsided after changing the time of administration to after meals or without intervention. There were no co-morbidities in the patient who discontinued treatment that could account for severe fatigue. These data are retrospective and non-randomized.
  • the ARMD in these eyes was not graded, and neither patients nor researchers were masked to the type of treatment received, which could potentially introduce a placebo-effect bias.
  • VPA provides an effective treatment for human subjections with Retinitis Pigmentosa
  • VPA Valproic Acid
  • RP Retinitis Pigmentosa
  • Table 3 summarizes the average characteristics of the RP patients included in this analysis.
  • Retinitis Pigmentosa is a blinding disease with no robust treatment options.
  • the visual field areas of five of seven RP patients increased with a short treatment of valproic acid. Encouragingly, in one case(patient # 6), the significant improvement in functioning retinal area was confirmed at two time points (23 and 27 weeks). While visual acuity is not always a reliable outcome measure for RP given that
  • Valproic acid is widely used as an anti-convulsant and mood stabilizer and its efficacy in these capacities is likely mediated via its ability to affect GABA levels through glutamic acid decarboxylase and GABA transaminase modulation.
  • VPA was identified using a heterologous cell culture screen for small molecules that increase the yield of properly folded RP mutant rhodopsins. Without wishing to be bound by theory, a variety of evidence indicates that VPA likely works at the level of cell death protection or inflammatory mediation as its neuroprotective properties have been well documented (Feng et al., Neuroscience. 2008; 155:567-72; Leng et al., J Neurosci.
  • VPA is known to be a potent inhibitor of histone deacetylase (HDAC) (Gottiere et al., Valproic acid defines a novel class of HDAC inhibitors inducing differentiation of transformed cells. EMBO J. 2001 ;20:6969-78).
  • HDAC histone deacetylase
  • VPA can induce cells to differentiate in culture (Gottiere et al., Valproic acid defines a novel class of HDAC inhibitors inducing differentiation of transformed cells. EMBO J. 2001 ;20:6969-78); moreover, it has been shown to stimulate glial cells to differentiate into photoreceptor-like cells.
  • VPA provides a safe and effective therapy for RP, a tragic blinding disease for which no effective therapies currently exist.
  • the results of the clinical analysis reported herein in conjunction with the in vitro data provided above indicate that VPA is an effective treatment for photoreceptor loss associated with RP.
  • This study provides the basis for a placebo-controlled clinical trial with patients with well characterized RP genotypes to more fully evaluate the efficacy and safety of VPA as a treatment for RP.
  • ARPE- 19 cells were obtained from the American Type Culture Collection (ATCC CRL2302, Manassas, VA) and grown in high glucose Dulbecco's Modified Eagle Medium (DMEM, Cellgro/Mediatech Inc., Manassas, VA) supplemented with 10% heat-inactivated fetal calf serum (FCS, Sigma-Aldrich, St.Louis, MO) and 1 % penicillin/streptomycin (Gibco, Grand Island, NY) at 37°C in presence of 5% C0 2 . Cells were routinely subcultured or harvested for experiments using Tryple Express (Gibco). In all subsequent experiments, cells were grown from frozen aliquots to ensure that all experiments were conducted on similar cells between passage (P)6 to P9.
  • HSF1 knockout mouse embryonic fibroblasts referred to as MEF 4" ' "1" and MEF A respectively, were generously provided by Dr. Benjamin's lab (McMillan et al., The Journal of Biological Chemistry, 273, 7523-7528).
  • MEF's were cultured in high glucose DMEM supplemented with 0.1 mM non-essential amino acids (Gibco), 1 % penicillin/streptomycin, 0.1 mM ⁇ -mercaptoethanol (Sigma), and 10% FCS at 37°C in presence of 5% C0 2 .
  • ARPE- 19 and MEF cells were grown to confluency on 100mm plates before being subjected to hydroquinone treatment at various concentrations for 48 hours or 72 hours in the presence of valproic acid. After addition of hydroquinone, the media and PBS wash were collected and spun at 3500 rpm for 5 min along with the trypsinized cells. The cell pellet was resuspended in ice-cold PBS and cell viability was evaluated by the Live/Dead mammalian cell viability kit (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol.
  • Live/Dead mammalian cell viability kit Invitrogen, Carlsbad, CA
  • cell number was adjusted to 1 x 10 6 /mL PBS before adding calcein AM and ethidium homodimer and after 15 minutes incubation at room temperature in the dark; the samples were analyzed by FACSCalibur (BD Biosciences, San Jose, CA). Ubiquitous esterase activity within cells was required to generate the calcein AM fluorescence while dead cells with compromised membranes were labeled by ethidium homodimer. Results are presented as a percentage of total cellular fractions stained with the appropriate fluorophore. Annexin V labeling
  • ARPE- 19 and MEF cells were grown, treated, and collected as previously described (McMillan et al., The Journal of Biological Chemistry, 273, 7523-7528) and were then subjected to Annexin V-fluorescein isothiocyanate (AV-FITC) staining (Calbiochem, San Diego, CA) according to the manufacturer's instructions. Briefly, the cell pellet was re-suspended in fresh media, with media-binding reagent added to the suspension followed by Annexin V-FITC. After a 15 minute dark incubation at room temperature, the cells were spun at 1000 x g for 5 minutes.
  • AV-FITC Annexin V-fluorescein isothiocyanate
  • the pellet was resuspended in cold I X binding buffer and then propidium iodide (PI) was added.
  • PI propidium iodide
  • the samples were analyzed by FACSCalibur (BD Biosciences) and a portion of the suspension was also placed on slides for immediate observation using a Leica
  • ARPE- 19 and MEF cells were grown, treated, and collected as described above and were then subjected to cleaved caspase-3 staining (Calbiochem, San Diego, CA) according to the manufacturer's instructions.
  • the cell pellet was resuspended in ice-cold PBS and cell number was adjusted to 1 x 10 6 /mL.
  • 1 ⁇ _ of FITC-DEVD- FMK was added to 300 ⁇ _ of each sample which was then incubated in a 37°C incubator with 5.0% C0 2 for 60 minutes. The cells were then washed three times by centrifugation at 3000 rpm for 5 minutes before being re-suspended in wash buffer.
  • Negative controls were treated with a caspase-3 inhibitor (Z-VAD-FMK) as instructed by the kit.
  • the marker indicates percentage of cells with greater than a predetermined level of fluorescence from control samples indicated as cells positive for cleaved caspase-3 by the kit.
  • ARPE- 19 cells were grown on sterile coverslips to about 80-90% confluency before drug/stressor additions as described above. After 72 hours, the media was removed, the cells were washed with PBS before immediate addition of 4% paraformaldehyde in PBS for 15 minutes at room temperature. After washing the fixed cells with PBS, -20°C methanol was added for 10 minutes. Blocking was accomplished by addition of 5% normal serum (Sigma-Aldrich) from the same species that the secondary antibody is derived from. After 60 minutes blocking at room temperature, a 1 :200 dilution of mouse-derived anti cleaved caspase-3 antibody (Cell Signal, Danvers, MA) was added to the coverslips.
  • coverslips were washed three times in PBS before being incubated in 1 :200 anti-mouse FITC antibody (Jackson Immunoresearch, West Grove, PA) for 90 min. After three 5 minute washes with PBS, the coverslips were mounted to slides with anti-fade Vectashield (Vector Labs, Burlingame, CA) containing 4',6-diamidino- 2-phenylindole (DAPI). Slides were then viewed by a Leica DMI6000B fluorescence microscope and images were acquired by Image ProPlus (Media Cybernetics, Bethesda, MD). TUNEL Staining
  • ARPE- 19 cells were grown, and treated on sterile coverslips as described above.
  • the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining (Roche, Indianapolis, IN) was carried out according to manufacturers instructions. After treatment, the media was removed and the cells were washed with PBS before immediate addition of 4% paraformaldehyde in PBS for 60 minutes. After rinsing with PBS, the coverslips were then incubated in 0.1 % Triton X- 100 in 0.1 % sodium citrate for 2 minutes at 4°C. Coverslips were rinsed twice with PBS and were then incubated with the TUNEL reaction mixture for 60 minutes at 37°C.
  • TUNEL terminal deoxynucleotidyl transferase dUTP nick end labeling
  • MEF*'* and MEF A cells were grown to confluency on 100mm plates before incubation with valproic acid. The cells were then enzymatically detached and pelleted after washing with PBS. The pellet was then lysed (0.1 % dodecyl maltoside, IX protease inhibitor cocktail (Roche)) for 60 minutes at 4°C on a tube rotator. The suspension was then spun at 13000 rpm for 10 min at 4°C and the resulting supernatant was mixed with an equal volume of 2X lamelli SDS-PAGE buffer (Invitrogen).
  • Human fetal eyes ( 16- 18 weeks gestation) were obtained from Advanced Bioscience Resources (Alameda, CA). Primary cell cultures of hfRPE cells were prepared from human fetal eyes and cultured as described previously. Cells for fluid transport experiments (Jv) were cultured in transwells to confluence for 3 - 4 weeks for pigmentation and total tissue resistance (RT) ⁇ 400 ⁇ -cm 2 . For Jv measurements, a modified Ossing chamber was used to mount confluent monolayers of hfRPE using MEM-alpha (Sigma M4526) media and a capacitance probe technique as previously described.
  • MEM-alpha Sigma M4526
  • a nylon mesh supporting hfRPE was placed in a Kel-F chip in the fluid transport apparatus, and a capacitance probe measured changes in liquid level height as water moved across the RPE.
  • Transepithelial potential (TEP) was measured using Ag/AgCl pellet electrodes.
  • Valproic acid was perfused into both apical and basal bathing solutions of the hfRPE in untreated (control) transwells or in 24 hour valproic acid pre-treated ( 1 ⁇ and 5 ⁇ ) transwells and the recordings continued for another 30-60 minutes until steady state Jv was measured.
  • Valpromide 100 ⁇ , 400 ⁇ , 800 ⁇ was perfused into both apical and basal solutions of the hfRPE and Jv, TEP and RT were measured as a control.
  • IOP intraocular pressure
  • BCVA best-corrected visual acuity
  • OCT optical coherence tomography
  • logMAR logarithm of minimum angle of resolution
  • the change in visual field (mm 2 ) was defined as a simple measure of percent change from baseline:
  • VF areas (mm units per year ( 16). VF areas (mm ) were log g transformed (logVF) and the difference between follow-up and baseline was calculated (AlogVF). To calculate average percent change in VF over the course of treatment the average
  • Visual acuity was measured using a Snellen chart at a distance of twenty feet.

Landscapes

  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Chemical & Material Sciences (AREA)
  • Public Health (AREA)
  • Epidemiology (AREA)
  • Inorganic Chemistry (AREA)
  • Ophthalmology & Optometry (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne des compositions oculaires qui contiennent de l'acide valproïque et des analogues de l'acide valproïque, et des procédés d'utilisation de telles compositions en vue de réduire la mort de cellules oculaires (p. ex. la mort de cellules de l'épithélium pigmentaire de la rétine), en particulier la mort de cellules associées à une dégénérescence maculaire liée à l'âge.
PCT/US2011/023925 2010-02-05 2011-02-07 Compositions et procédés pour traiter ou prévenir une dégénérescence de la rétine Ceased WO2011097577A2 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US30174810P 2010-02-05 2010-02-05
US61/301,748 2010-02-05
US35071110P 2010-06-02 2010-06-02
US61/350,711 2010-06-02

Publications (2)

Publication Number Publication Date
WO2011097577A2 true WO2011097577A2 (fr) 2011-08-11
WO2011097577A9 WO2011097577A9 (fr) 2011-12-22

Family

ID=44356105

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2011/023925 Ceased WO2011097577A2 (fr) 2010-02-05 2011-02-07 Compositions et procédés pour traiter ou prévenir une dégénérescence de la rétine

Country Status (1)

Country Link
WO (1) WO2011097577A2 (fr)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3222324A1 (fr) * 2016-03-23 2017-09-27 Wayne State University Valproate en tant que traitement antifongique topique
CN108251371A (zh) * 2018-01-11 2018-07-06 苏州市立医院（苏州市妇幼保健院、苏州市中心体检站、苏州市公惠医院、苏州市立医院司法鉴定所、苏州市肿瘤诊疗中心） 视网膜色素上皮细胞体外培养方法及其专用培养基
CN109633910A (zh) * 2019-01-14 2019-04-16 京东方科技集团股份有限公司 Ar/vr隐形眼镜及其制作方法和电子设备
WO2019240946A1 (fr) 2018-06-11 2019-12-19 The Regents Of The University Of California Déméthylation pour traiter une maladie oculaire
US20220397959A1 (en) * 2014-11-10 2022-12-15 Irisvision, Inc. Methods and Systems for Enabling the Remote Testing of Vision and Diagnosis of Vision-Related Issues
RU2804300C2 (ru) * 2018-06-11 2023-09-27 Дзе Риджентс Оф Дзе Юниверсити Оф Калифорния Деметилирование для лечения глазного заболевания

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20220397959A1 (en) * 2014-11-10 2022-12-15 Irisvision, Inc. Methods and Systems for Enabling the Remote Testing of Vision and Diagnosis of Vision-Related Issues
EP3222324A1 (fr) * 2016-03-23 2017-09-27 Wayne State University Valproate en tant que traitement antifongique topique
US10092531B2 (en) 2016-03-23 2018-10-09 Wayne State University Valproate as a topical anti-fungal treatment
US10617659B2 (en) 2016-03-23 2020-04-14 Wayne State University Valproate as a topical anti-fungal treatment
CN108251371A (zh) * 2018-01-11 2018-07-06 苏州市立医院（苏州市妇幼保健院、苏州市中心体检站、苏州市公惠医院、苏州市立医院司法鉴定所、苏州市肿瘤诊疗中心） 视网膜色素上皮细胞体外培养方法及其专用培养基
WO2019240946A1 (fr) 2018-06-11 2019-12-19 The Regents Of The University Of California Déméthylation pour traiter une maladie oculaire
KR20210020008A (ko) * 2018-06-11 2021-02-23 더 리젠츠 오브 더 유니버시티 오브 캘리포니아 눈 질환을 치료하는 탈메틸화
EP3801465A4 (fr) * 2018-06-11 2022-03-23 The Regents of the University of California Déméthylation pour traiter une maladie oculaire
RU2804300C2 (ru) * 2018-06-11 2023-09-27 Дзе Риджентс Оф Дзе Юниверсити Оф Калифорния Деметилирование для лечения глазного заболевания
KR102785135B1 (ko) * 2018-06-11 2025-03-21 더 리젠츠 오브 더 유니버시티 오브 캘리포니아 눈 질환을 치료하는 탈메틸화
CN109633910A (zh) * 2019-01-14 2019-04-16 京东方科技集团股份有限公司 Ar/vr隐形眼镜及其制作方法和电子设备
CN109633910B (zh) * 2019-01-14 2021-11-05 京东方科技集团股份有限公司 Ar/vr隐形眼镜及其制作方法和电子设备

Also Published As

Publication number Publication date
WO2011097577A9 (fr) 2011-12-22

Similar Documents

Publication Publication Date Title
AU2009240470B2 (en) Inhibition of neovascularization by cerium oxide nanoparticles
US10966962B2 (en) Method for treating neurodegenerative diseases
US20100087474A1 (en) Materials and methods for enhanced degradation of mutant proteins associated with human disease
JP2019034947A (ja) 眼疾患を予防または治療するための方法および組成物
KR20080033463A (ko) 단백질 미스폴딩을 보정하는 작은 화합물 및 그의 용도
CN106999543B (zh) 包含环孢霉素和海藻糖的眼用组合物
WO2019133904A9 (fr) Inhibiteurs des voies de hsp90, de la pi3-kinase, du protéasome, de la hdac et de p97 pour l'élimination sélective de cellules sénescentes dans le traitement de conditions liés à l'âge
CN110869045B (zh) 包含i型胶原及色素上皮衍生因子肽作为有效成分的预防或治疗新生血管性疾病的药物组合物
WO2011097577A2 (fr) Compositions et procédés pour traiter ou prévenir une dégénérescence de la rétine
CN109789182A (zh) 治疗干眼综合征的方法
JP2018530586A (ja) 加齢黄斑変性のための高用量スタチン
CA2819616A1 (fr) Procedes de traitement de maladies de la retine
JP2017533175A (ja) 角膜ジストロフィーの予防および／または治療のためのサフランをベースとした組成物
EP3548504A1 (fr) Inhibiteurs de protéasome à base de peptides pour le traitement d'affections induites par des cellules sénescentes et pour le traitement du cancer
EP3875092B1 (fr) Composition de prévention ou de traitement de la dégénérescence maculaire
JP2022531484A (ja) 加齢性黄斑変性症を治療するための物質および方法
Mitter et al. Autophagy in ocular pathophysiology
CN101287370A (zh) 用于增强与人类疾病相关的突变蛋白质的降解的物质与方法
JP6764233B2 (ja) 眼疾患処置薬
CN119300821A (zh) 利福霉素的眼用组合物及其用途

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 11740496

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase in:

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 11740496

Country of ref document: EP

Kind code of ref document: A2